HIGH DOSE PEMETREXED WITH LEUCOVORIN RESCUE Mathew A Cherian

1. HIGH DOSE PEMETREXED WITH LEUCOVORIN RESCUEMathew A Cherian

2. HIGH DOSE ANTI-FOLATE THERAPY

3. Methotrexate:History Aminopterin was the first drug reported to cause remissions in children with acute lymphoblastic Leukemia, and Methotrexate, was the first drug that was shown to be curative for patients with a solid tumour, Choriocarcinoma. 10 years after was introduced in the clinic, the mechanism of the action of as a potent inhibitor of dihydrofolate reductase was established by Osborn, Freeman and Huennekens ( Proc Soc Exp Biol Med 97(1958) pp 429-451)

5. Mechanism of Antifolates

6. The de novo purine biosynthetic pathway. It is a ten-step pathway involving the conversion of phosphoribosylpyrophosphate to inosine monophosphate. All substrates and intermediates in the pathway are labeled. The third step is catalyzed by the enzyme glycinamide ribonucleotide transformylase (GAR Tfase). This enzyme is monofunctional in E. coli, but exists as part of a trifunctional protein in mammalian cells. The three activities that are condensed are GAR Tfase, GAR synthase, and AIR synthase. The latter enzymes catalyze step 2 and step 5 of purine biosynthesis. Aminoimidazole ribonucleotide transformylase (AICAR Tfase) catalyzes step 9 and exists as a bifunctional, condensed with the tenth activity of the pathway inosine monophosphate (IMP) cyclohydrolase from E. coli to humans. AICAR Tfase uses the same 10-formyltetrahydrofolate cofactor as GAR Tfase.The de novo purine biosynthetic pathway. It is a ten-step pathway involving the conversion of phosphoribosylpyrophosphate to inosine monophosphate. All substrates and intermediates in the pathway are labeled. The third step is catalyzed by the enzyme glycinamide ribonucleotide transformylase (GAR Tfase). This enzyme is monofunctional in E. coli, but exists as part of a trifunctional protein in mammalian cells. The three activities that are condensed are GAR Tfase, GAR synthase, and AIR synthase. The latter enzymes catalyze step 2 and step 5 of purine biosynthesis. Aminoimidazole ribonucleotide transformylase (AICAR Tfase) catalyzes step 9 and exists as a bifunctional, condensed with the tenth activity of the pathway inosine monophosphate (IMP) cyclohydrolase from E. coli to humans. AICAR Tfase uses the same 10-formyltetrahydrofolate cofactor as GAR Tfase.

7. MTX and Pemetrexed work by inhibiting both de novo pyrimidine AND purine synthesis

8. Polyglutamylation of antifolates is a key process

9. Polyglutamylation of antifolates is a key process Polyglutamylation results in enormous changes in the affinity of antifolates for thymidylate synthetase, amino imidazole carboxamide ribonucleotide transformylase and glycinamide ribonucleotide transformylase. Polyglutamylation markedly prolongs the duration of action of antifolates by trapping them intracellularily:Polyglutamates of THF coenzymes and antifolates are polyanions which are impermeable to lipid bilayers and are hence retained intracellularly.

10. Polyglutamylation of antifolates is a key process Relatively long chain (n>3) polyglutamate derivatives of THF coenzymes and antifolates are no longer substrates of membrane transport systems including folate influx transporters (including RFC) and efflux transporters such as multidrug resistance proteins breast cancer resistance protein (BCRP). The level and time-course of formation of antifolate polyglutamates in tumor cells is a key determinant of the cytotoxic activity exerted by folate antimetabolites.

11. Mechanisms of resistance to Methotrexate Increased DHFR expression (transcriptional or gene amplification). Decreased affinity of DHFR for MTX Alterations in membrane transport Efflux transporters Impaired polyglutamylation Many of these can be overcome by high doses of MTX.

12. High dose Methotrexate therapy Goldin developed the concept of rescuing normal cells from toxicity by providing reduced folates (folinic acid, N5-formyl-tetrahydrofolate, citrovorum factor) to bypass the metabolic block induced by MTX . In these pioneering experiments, administration of leucovorin within 24 to 36 hours after administration of MTX was able to prevent MTX-induced host toxicity without diminishing antitumor activity. If administered correctly high dose methotrexate causes no cytopenias or mucositis.

13. Applications of High Dose Methotrexate Osteosarcoma Primary CNS lymphoma Secondary CNS involvement by NHL Leptomeningeal metastasis Lymphomatous meningitis ALL Burkitts Lymphoma

14. Mechanisms of High Dose Methotrexate By achieving higher extracellular drug concentrations, HDMTX may facilitate entry of MTX into cells; this may be of particular importance in the eradication of clones resistant to MTX on the basis of cell membrane transport defects. The second advantage is enhanced MTX-PG formation with HDMTX therapy may be facilitated by exposure to higher extracellular MTX concentrations as well as prolonged exposure. High blood levels of MTX overcome a variety of mechanisms of resistance (i.e. decreased affinity for RFC, DHFR, FPGS) and generate high free intracellular levels through passive diffusion. The ability to eradicate resistant clones that contain greater concentrations of DHFR as a result of enhanced transcription or gene amplification may also be critical.

15. Mechanism of the leucovorin rescue Phenomenon:why are only normal cells rescued? There are substantial data to support the concept that the biochemical basis for the selectivity of leucovorin rescue is related to a greater formation of MTX polyglutamates in tumor versus host cells and the ability of these congeners to block the utilization of added folates. In normal regenerating cells of the bone marrow and GI there is only low-level formation of MTX polyglutamates (Poser et al., 1981; Fry et al., 1983; Fabre et al., 1984; Koizumi et al., 1985). In tumor cells that accumulate high levels of MTX polyglutamates, rescue is impaired because there is direct suppression by these congeners of the utilization of the added folates at the level of the THF-cofactor-requiring enzymes (Matherly et al., 1987; Koizumi et al., 1990). . Tumours may accumulate polyglumates because they start off with smaller intracellular pools of folate.

16. Best Pract Res Clin Haematol. 2009 Dec;22(4):577-82.

17. Folate and Anti-Folate Transport RFC(Reduced Folate Carrier) FR a (Folate receptor a) FR ß (Folate receptor ß) PCFT(Proton Coupled Folate Transporter) Passive diffusion(only at >100µM)

18. Folate and AntifolateTransport Reduced folate carrier is a major transporter of antifolate drugs used for cancer chemotherapy such as methotrexate, pemetrexed, and raltitrexed( J Biol Chem. 2009 Jan 30;284(5):3285-93. )  Folate receptors are believed to be a useful biological target for disease management, because its tissue expression profile appears to be limited to tissues that are responsible for whole body retention of folates (eg, the placenta, choroid plexus and kidney) and, more importantly, to certain pathological tissues, such as tumors and sites of chronic inflammation.(Cancer Metastasis Rev. 2008 Dec;27(4):655-64.) Proton Coupled Folate Transporter: When the pH is 7.4, RFC activity is optimal and (PCFT) activity is minimized. Hence, under these conditions, transport of antifolates will be mediated predominantly by RFC. On the other hand, as the pH is decreased this relationship reverses so that at the pH of solid tumors the role of PCFT becomes more prominent. Pemetrexed has the highest affinity for the low-pH transporter among all the folates and antifolates that have been studied.(Cancer Metastasis Rev (2007) 26:129–139)

20. Pemetrexed: a Superior Antifolate

21. Pemetrexed: a Superior Antifolate

22. Comparison of pemetrexed and methotrexate polyglutamylation kinetics. Pemetrexed has a far lower Michaelis constant for human recombinant FPGS than does methotrexate. Experimental techniques are detailed in ref. (137).Comparison of pemetrexed and methotrexate polyglutamylation kinetics. Pemetrexed has a far lower Michaelis constant for human recombinant FPGS than does methotrexate. Experimental techniques are detailed in ref. (137).

23. Pemetrexed: a Superior Antifolate In HeLa cells, loss of the RFC gene results in the preservation of pemetrexed growth inhibition in medium in which 5-CHOTHF is the source of intracellular folates. This was attributed to a substantial level of residual pemetrexed transport mediated by an RFC-independent process and a substantial depletion of cellular Tetrahydrofolate-cofactors when RFC function is lost due to impaired transport of 5-CHO-THF, resulting in partial preservation of pemetrexed polyglutamylation, compensating in part for the loss of transport, and increased inhibition at the level of GARFT . In the HCT-15 colon carcinoma cell line, loss of RFC function results in a marked reduction in pemetrexed transport with only a low level (15%) of residual pemetrexed influx. This is associated with marked contraction of cellular pools of reduced folates when cells are grown in 5-CHO-THF, and 3-fold increased sensitivity to pemetrexed. Transfection of RFC back into PT1 cells(HCT-15 subline lacking RFC) increased sensitivity to methotrexate and decreased sensitivity to pemetrexed to levels observed in wild-type cells. This suggests that loss of RFC as a mechanism of resistance to Pemetrexed is not an option for cancer cells as the loss of intracelular folate pool leads to enhanced retentionof Pemetrexed polyglutamates which likely more than compensateds for decreased influx.

24. Pemetrexed cytotoxicity is less sensitive to duration of cell exposure

25. Folates antagonize antifolate activity Increased density of FR in tumors implanted in low folate diet mice and a decrease in the affinity of these receptors for folic acid. The results suggest that tumors can compensate for low folate bioavailability by up-regulation of a second FR with slightly lower affinity for folic acid. Changes in dietary folate intake can modulate FPGS activity significantly in vivo and suggest that the tissue distribution and toxicities of classical antifolates requiring polyglutamation for activation and cellular retention will be influenced significantly by folate status of the host.

26. Antagonism of Antifolates by Leucovorin

27. Antagonism of Antifolates by Leucovorin Dependence of antifolate growth inhibition upon extracellular 5-CHO-THF with continuous exposure to drugs. L1210 cells grown in 96-well plates in medium containing different concentrations of 5-CHO-THF were exposed continuously to a range of drug concentrations for 3 days. Fold change in 50 on the ordinate is the ratio of 50 values at the indicated extracellular 5-CHO-THF level to the 50 values at the lowest 5-CHO-THF concentration (0.64 nM)

29. Pharmacokinetics Pharmacokinetic evaluations have shown that pemetrexed exhibits dose-proportional pharmacokinetics over a broad dose range Physicians' Desk Reference. Thomson PDR, Montvale, NJ. Alimta, pemetrexed for injection, 2006 edition

30. Pharmacokinetics of Pemtrexed vs Methotrexate

31. Solubility of Methotrexate versus Pemetrexed Methotrexate solubility: practically insoluble in water, but soluble in alkaline solution. At a urine pH of 5 conc>1 mmol/L(0.45 mg/ml) exceeds the solubility and can lead to acute renal failure due to crystal nephropathy. At a pH of 6 the solubility of methotrexate is 1.55 mg/ml and 9 mg/ml at a pH of 7. Pemetrexed solubility: freely soluble in water,90 mg /ml(10-100 times MTX) at pH 6-10. If given over 24 hrs with an average urine output of only 2 litres/day,purely from the point of view of solubility, a dose of 90 grams/m2 could be delivered..

32. Rationale for Clinical Trial Summarizing theprevious statements: 1)Pemetrexed may be a superior antifolate in terms of its versatile and higher affinity binding to cellular transporters and ability to effect a response, especially in solid tumours. 2)Very importantly Pemetrexed possesses a much lower Km and higher Vmax for polyglutamylation, a process which is key for selective rescue of normal cells vs tumour cells. 3)Very importantly Pemetrexed possesses free solubility in water from pH 6 to 10. 4)Pemetrexed is effectively antagonized by leucovorin. 5)Pemetrexed possesses slightly better CNS penetration Putting these together a phase I dose escalation trial of high dose Pemetrexed with leucovorin rescue is warranted.

33. Proposed Clinical Trial:High Dose Pemetrexed with 5-Formyl tetrahydrofolate(Citrovorum Factor) Rescue Target patient population: 1. Malignant solid tumours with small asymptomatic brain metastases before or after stereotactic radiation therapy. 2. Malignant solid tumours with brain metastases after relapse following whole brain radiation therapy. 3. Malignant solid tumours with leptomeningeal metastases relapsing after or following first line therapy. 3. Relapsed Glioblastoma Multiforme and other primary CNS neoplasms including relapsed primary CNS lymphoma. 4. Relapsed low, intermediate and high grade lymphomas with CNS involvement. 5. Relapsed ALL and AML following 2nd line therapy

35. Choosing the correct dose of leucovorin

36. Currently Active Trials Phase I trials of conventional schedules of Pemetrexed are already underway at other institutions in GBM and Primary Central Nervous System Lymphoma. However these trials use conventional folate and B12 supplementation prior to infusions and attempt dose escalation.

37. Thank you!